Esterases and Lipases.
Esterases and lipases catalyze the hydrolysis of ester bonds to produce alcohols and carboxylic acids as shown below. ##STR1##
Esterases and lipases can be characterized by different substrate specificities, R group or chain length preference, and unique inhibitors (1, 2). The many esterases and lipases range from hydrolases such as the broad carboxyl esterases which preferentially hydrolyze esters with long carbon chain R groups, to choline esterases, and to acetyl esterases which act on very specific substrates. In many cases, these hydrolases are also known to show stereo- and regio-selective preferences resulting from the chiral nature inherent in protein active sites. This preferential hydrolytic activity make them useful for reactions requiring different regioselectivity and stereoselectivity or for kinetic resolution methods on racemic mixtures. For enzymes that demonstrate stereoselectivity, if R* is a racemic mixture, the product of enzyme catalyzed hydrolysis, R.sub.1, would be the most rapidly hydrolyzed stereoisomer while the remaining ester designated R*' would be the enriched antipode mixed with any remaining R.sub.1. The products can then be separated by chromatography to provide pure R.sub.1. The availability of a large pool of esterases and lipases with varying specificities would be useful for screening the enzymes for specific reactions, and developing optimal protocols for specific chemical synthesis. The expedience of this process would facilitate the production scale-up of many useful pharmaceutical products.
In aqueous solvent systems, esterases and lipases carry out their natural reactions: the hydrolysis of ester bonds. In vitro, these enzymes can be used to carry out reactions on a wide variety of substrates, including esters containing cyclic and acyclic alcohols, mono- and di-esters, and lactams (3). By carrying out the reactions in organic solvents (4, 5) where water is excluded, the reactions of esterases and lipases can be reversed. These enzymes can catalyze esterification or acylation reactions to form ester bonds (3, 6, 7). This process can also be used in the transesterification of esters and in ring closure or opening reactions.
Optically pure chiral pharmaceuticals.
Currently, the majority of synthetic chiral pharmaceuticals are sold as racemic mixtures. However, due to advances in the synthesis of optically pure (single isomer) chiral compounds, this situation is changing (7). Racemic drugs often contain one isomer which is therapeutically active and the other enantiomer which is at best inactive and at worst a major cause of potentially harmful side effects. The non-useful isomer in a racemic drug is increasingly being viewed as a contaminant. Indeed, the FDA's Policy Statement for the Development of New Drugs recommends "that the pharmacokinetic profile of each isomer should be characterized in animals and later compared to the clinical pharmacokinetic profile obtained in Phase I" drug testing (8). Thus, pharmaceutical companies will need to develop a synthesis or separation route to produce each pure isomer of each new synthetic drug.
Enzymatic synthesis of optically pure pharmaceuticals and intermediates.
Since it is often very difficult to generate optically pure solutions of certain chiral molecules by classical chemical synthesis, new enzymatic biocatalysts will play a major role in this endeavor. In some cases, enzymes may be able to replace hazardous chemical synthesis procedures with more environmentally-friendly biological synthesis processes. It can also be much more cost effective to produce a pharmaceutical intermediate enzymatically if an enzyme can eliminate several chemical protection and deprotection steps at once (7). All six major classes of enzymes (oxidoreductases, transferases, hydrolases, lyases, isomerases, and ligases) have been useful in the synthesis of optically pure compounds as described in several detailed reviews (3, 7). The hydrolases have proven to be the most useful group of enzymes, due to the abundance of hydrolases, the information about them, their independence from cofactors, and the wide variety of substrates they can accept.
A survey of the literature shows many examples of mesophilic hydrolases particularly esterases and lipases used in chemical synthesis or chiral resolution. These include esterases from pig (9, 10) and horse (3) livers and a wide variety of lipases from Aspergillus sp. (11) Candida sp. (12-16), Pseudomonas sp., (17-19), Rhizopus sp. (20) and others. Several lipases have been used in the synthesis of propranolol (7), a beta-adrenergic blocking agent used in the treatment of angina and hypertension. Ibuprofen, a nonstearoidal antiinflammatory agent has been synthesized via stereo selective hydrolysis of its methyl ester using carboxyesterase (7). While these enzymes have begun to demonstrate the utility of biocatalysts in chemical synthesis, there is still a profound need for a wider variety of esterases and lipases which have varying substrate specificities, regioselectivities, and steroselectivities. In addition, since these enzymes need to be employed in a large-scale industrial setting, there is a need for them to have increased stability, higher thermotolerance and a longer "shelf life".
Thermostable enzymes.
Thermophilic organisms have already provided a rich source of useful proteins that catalyze reactions at higher temperatures and are stable for much longer periods of time (21, 22). One example is the DNA Polymerase I from Thermus aquaticus and its use in polymerase chain reaction (PCR) (23, 24). Thermophilic enzymes have become the most commercially successful enzymes in industry because of their long-term stability and ease of use. The most successful enzyme to date, alpha-amylase, is used in corn processing and comes from the moderate thermophile B. stearothermophilus (25). Another commercially successful industrial enzyme is subtilisin, a serine protease also found in various strains of Bacillus, has been widely used in laundry detergents and other cleaning solutions.
The commercial success of these enzymes can be attributed to their ease of use. In addition to functioning at high temperatures, thermostable enzymes generally posses an increased shelf life which markedly improves handling conditions, especially by those not trained in biochemistry to work with the specific range of conditions used for mesophilic enzymes. If enzymes are to play a significant role in large scale processing of chemicals, they must be able to endure the harsh conditions associated with these processes. Thermostable enzymes are easier to handle, last longer, and given the proper immobilization support should be reusable for multiple applications
Finally, the hydrophobic and electrostatic forces that allow these enzymes to survive high temperatures also allow them to generally function better in organic solvents (26-31). While most enzymes lose a significant portion of their activity in organic solvents, thermostable enzymes may prove more tolerant to the denaturing conditions of many organic solvents. Highly thermostable esterases and lipases are necessary to expand the application of these biocatalysts in large scale industrial reactions.
Thermostable esterases and lipases.
To date, only one esterase and a few lipases have been reported with moderately thermostable characteristics. Tulin et al. (32) reported a Bacillus stearothermophilus esterase cloned into Bacillus brevis which was stable up to 10 minutes at 70.degree. C. Sugihara et al.(33, 34) have isolated novel thermostable lipases from two microorganisms, A Bacillus soil isolate and a Pseudomonas cepacia soil isolate. The former lipase is stable up to 30 minutes at 65.degree. C. but rapidly inactivated above this temperature. The lipase from Pseudomonas cepacia was stable when heated for 30 minutes at 75.degree. C. and pH 6.5 but had only 10% of its activity when assayed at this temperature. A thermoalcalophilic lipase (35) was identified from a Bacillus species MC7 isolated by continuous culture and had a half-life of 3 hours at 70.degree. C. Finally, Sigurgisladottir et al. (6) have reported the isolation of one Thermus and two Bacillus strains which posses lipases active on olive oil up to 80.degree. C., although there was no report on enzyme stability in this study.
These enzymes offer only limited variations in substrate specificities and only moderate thermostability profiles. They do not address the need for different substrate specificities, the need to produce large scale quantities which can be economically commercialized, and many of them have only limited overall stability. In this patent application we have identified a series of esterases and lipases which offer a range of substrate specificities (including regioselectivity, stereoselectivity), enhanced enzyme stability, and can be produced in large quantities for commercial use.